Molecular Characterization of Hsp90 Isoforms in Colorectal Cancer Cells and Its Association with Tumour Progression

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Molecular characterization of hsp90 isoforms in colorectal cancer cells and its association with tumour progression

Z. MILICEVIC1, D. BOGOJEVIC2, M. MIHAILOVIC2, M. PETROVIC2 and Z. KRIVOKAPIC3

1Laboratory for Molecular Biology and Endocrinology, ‘Vinca’ Institute of Nuclear Sciences; 2Department of Molecular Biology, Institute for Biological Research; 3Institute for Digestive Diseases, Clinical Center of Serbia, Belgrade, Serbia

Received September 26, 2007; Accepted December 21, 2007

Abstract. A key role of hsp90 in the activity of various Introduction oncogenic proteins and pathways is currently of intense interest. To clarify the molecular basis of biological Hsp90 is of interest because of its importance in maintaining behaviour of colorectal cancers we analysed the expression the conformation, stability and function of key oncogenic characteristics of hsp90 in cytosolic, nuclear and plasma mem- proteins involved in signal transduction pathways leading to branous fractions of cancer cells. As determined by Western proliferation, cell cycle progression and apoptosis, as well as blot assay all hsp90 isoforms studied, α (84 kDa), ß (86 kDa) other features of the malignant phenotype such as invasion, and hsp90N (75 kDa), were up-regulated and differentially angiogenesis and metastasis (1). Molecular chaperones help expressed in various stages of colorectal carcinoma. The hundreds of signalling molecules to keep their activation- inducible hsp90α isoform is a component of invasive pheno- competent state, and regulate various signalling processes type of cancer cells thus pointing to the importance of hsp90α ranging from signalling at the plasma membrane to tran- for metastasis generation. The expression of hsp90ß is scription (2). Hsp90 participation in multimolecular complex definitely higher in poorly-differentiated carcinomas than in formation may be required for induction of the transformed well-differentiated cancers, suggesting an involvement of phenotype (3,4). Such a requirement would not be surprising hsp90ß in the inhibition of cancer cell differentiation. given that the assembly of multimolecular protein complexes Especially, the expression of cytosolic hsp90N isoform in has been shown to be a critical event in an increasing number malignant cells points to the possibility that induction or of signal transduction pathways regulating cell growth and overexpression of hsp90N might be causally related to differentiation, survival and tumour-specific transformation tumour formation. Hsp90N is the plasma-membrane- (5,6). associated protein in poorly-differentiated colorectal cancers Hsp90 is present in multiprotein machine complexes that with metastasis. This suggests that the expression of hsp90N are predominant in cancer cells but not normal cells. The is elevated with progressive dedifferentiation often associated presence of these hsp90 complexes in tumour cells is consistent with advanced cancer stages. Hsp90 was exclusively localized with a major role for hsp90 as the cancer chaperone-buffering in the invasive front in a majority of metastatic cancers as and stabilizing metastable and potentially damaging mutant visualized by immunohistochemical study. Consistent with proteins during the evolution and progression of malignancy these facts, the frequent expression of hsp90α and hsp90N on (7). This is consistent with the idea that hsp90 is absolutely the surface of colorectal cancer cells may enable hsp90 to act required for malignant progression (8). as a mediator of metastasis generation. The above results The hsp90 superfamily comprises the cytoplasmic indicate more complex roles for hsp90 in colorectal chaperones hsp90α and hsp90ß, together with the endo- tumourigenesis. In this way, the hsp90 would be at the plasmic reticulum homologue Grp94 and the mitochondrial crossroads of both signalling and cell migration events. homologue TRAP1 (9). Grp94 and TRAP1 are involved in chaperoning proteins in their respective subcellular com- partments, although the evidence for this is weak for TRAP1. ______Hsp90N, a variant of the cytoplasmic hsp90, has been previously identified and appears to be membrane associated (10). As described, hsp90N bound Raf-1 more tightly than Correspondence to: Dr Desanka Bogojevic, Institute for Biological hsp90 and it might recruit Raf-1 to the membrane (10). Raf-1 is Research, Blvd. Despot Stephan 142, Belgrade, Serbia E-mail: [email protected] a serine/threonine kinase which participates in an evolutionarily conserved signalling cascade transducing signals from the Key words: hsp90, colorectal cancer, malignant progression plasma membrane to the nucleus (11). Raf-1 plays critical roles in cell growth and differentiation and induces the expression of early response genes (12). Raf-1 kinase associates with the hsp90 chaperone complex containing hsp90N, leading to 1169-1178 7/5/08 18:26 Page 1170

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Raf-1 stabilization and inhibiting its proteasome-dependent X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF) and degradation (13). 50 mM Tris-HCl (pH 7.4), followed by centrifugation of the The hsp90 chaperones are also of special relevance in homogenate at 800 x g for 10 min. The supernatants were human cancer among proteins inhibiting apoptosis fulfilling spun at 11000 x g at 4˚C for 25 min and the resulting the criteria of anti-apoptotic members (14-17). There is also supernatant was again centrifugated at 20000 x g at 4˚C for evidence that tumour cells with high MHC class I expression 45 min. The pellet, containing plasma membranes and and susceptibility to CTL action may escape apoptosis by a associated proteins, was resuspended in 10 mM Tris-HCl mechanism which involves increased nuclear hsp90 (18). (pH 7.4) and stored at -20˚C. The cytosolic fraction was The question of how molecular chaperones function at key obtained by further centrifugation of supernatant at 100000 x g, regulatory points in the control of apoptosis is currently of at 4˚C for 60 min. The pellet obtained after first centrifugation intense interest. As a result of this the overexpression of was homogenized in ice-cold buffer (2.2 M sucrose, 5 mM

hsp90 is common in various human tumours, preferentially MgCl2, 0.5% Triton X-100, 1 mM PMSF and 50 mM Tris-HCl, in malignant cancers, correlates with poor prognosis and pH 7.4) and further purified by centrifugation. After this step resistance to therapy (19). the pellets representing the soluble nuclear proteins were

A further study (20) demonstrated that hsp90 play resuspended in 0.25 M sucrose, 5 mM MgCl2, 0.5% Triton important roles in cell differentiation through the control of X-100, 1 mM PMSF, and 50 mM Tris-HCl, pH 7.4. The tyrosine kinase activities. Hsp90 has multiple cellular roles, protein concentration was determined by the Lowry method many of which are poorly understood (21). (25) and samples were aliquoted and stored at -20˚C. The We focused on hsp90α/ß, which are the best understood presence and purity of plasma membranes isolated from at the present time. Furthermore, hsp90N expression was human colorectal cancer tissues and normal mucosa was analysed in colorectal cancer. The protein called hsp90N examined by membranous NaK-ATPase activity, according (75 kDa) is thought to have more specialized complex to the methods described earlier (26,27). ATPase activity was activities in the cell. In this instance, a strong correlation has measured by colorimetric determination of inorganic been suggested between higher levels of hsp90N expression phosphate (26), liberated from ATP. Incubation was carried and neoplastic transformation, including interruption of gap out at 37˚C for 10 min in a medium containing (in mmol/

junctional, intercellular communication and anchorage- l):50 Tris-HCl, pH 7.6, 1 EGTA, 5 MgCl2, 1 ATP and 20 μg independent proliferation (10). of membrane preparation in a final volume of 200 μl. Only the samples which displayed NaK-ATPase activity were Materials and methods considered as pure and taken for further analyses.

Subjects. Forty-eight surgically removed colorectal carcinoma Western blot analysis. Proteins isolated from normal mucosa and 12 normal colonic and rectal mucosal samples were and colorectal cancer cells were suspended in sample loading examined. Thirty-two patients were male, and 16 were female. buffer (0.1 M Tris, pH 6.8, 20% glycerol, 4% SDS, 0.04% The mean age of the patients was 62 years (range: 33-80). bromophenolblue, 10% ß-mercaptoethanol), and separated The primary lesion was located in the rectum in 32, the sigmoid electrophoretically on 12% acrylamide gels, according to the colon in 8, the descending colon in 4, the transverse colon in 2, procedure of Laemmli (28). Approximately 50 μg of nuclear, the ascending colon in 1, and the cecum in 1 of the patients. 20 μg of cytosolic and 80 μg of plasma membrane proteins The histological classification of the tumours was established per lane was loaded and run on 12% SDS-PAGE. Separated according to previous criteria (22). Thus, 20 were well differ- proteins were transferred onto nitrocellulose membrane entiated adenocarcinoma, 10 were moderately differentiated (Hybond, Amersham) according to the procedure of Towbin adenocarcinomas, and 18 were poorly differentiated adeno- et al (29). Transfer of proteins from the acrylamide gel to carcinomas. All cancers were graded according to the Astler- nitrocellulose was performed in transfer buffer (25 mM Tris, Coller's modified Dukes' stage as A, B1, B2, C1 and C2. The 192 mM glycine, 0.1% SDS, 20% methanol, pH 7.5) at 30 V, distribution of these tumours according to Astler-Coller system 40 mA overnight at 4˚C. Nitrocellulose filters were blocked was stage B1, 6 cases, stage B2, 26 cases and stage C2, 16 overnight with blocking buffer (1% BSA in 50 mM Tris, cases. Twelve normal colonic and rectal mucosal samples were 0.9% NaCl, 0.05% Tween-20, pH 7.5). The filters were then selected from macroscopically normal areas; 6 samples were incubated for 1 h 45 min in buffer (50 mM Tris, 0.9% NaCl, taken from the areas >10 cm away from tumours, and 6 from 0.05% Tween-20, pH 7.5) containing the mouse monoclonal normal mucosa of non-tumour patients. All of the samples were antibodies at the indicated dilution (AC88 1:1000; H9010 collected at the time of surgery, snap-frozen in liquid nitrogen, 1:500). After 1 h of incubation with the AP-conjugated goat and stored at -80˚C. anti-mouse IgG (H+L) antibodies (ICN Pharmaceuticals) (diluted 1:1000) the proteins bands were visualized in a Tissue and cellular fractionation. The normal mucosa and alkaline phosphatase substrate 5-bromo-4-chloro-3-indolyl- colorectal cancer tissues were surgically dissected and frozen. phosphate and nitroblue tetrazolium (NBT/BCIP) (Gibco). The nuclear fraction of normal and tumour tissues were Scanning densitometry was used to determine peak areas to obtained by differential centrifugation (23), and the plasma assess the relative amount of protein in immunoreactive bands. membrane fraction was obtained by a modified method of The bands representing the hsp90 immunoreactivity were Bauer and Hurtenbach (24). Briefly, the samples of normal scanned by a computer-based laser densitometer (Pharmacia and neoplastic tissue were minced by fine dissection and Ultra Scan XL). The duplicate gel was directly stained with

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Figure 2. Immunohistochemical staining of hsp90 in normal and tumour tissues. (a) A weakly hsp90 immunoreactivity is seen in the cells of normal rectal mucosae. (b) Human breast cancer exhibit strong hsp90-positive

staining. (c) Invasive well-differentiated rectal cancer stage G1C1. Note the strong expression of hsp90 in the cytoplasm and on the surface of tumour cells. The number of positive cells is increased and staining intensity is greater. Avidin-biotin-peroxidase complex (ABC) method, counterstained Figure 1. Immunohistochemical staining of hsp90 in (a) well-differentiated with methyl green.

low-grade tumour stage G1B1 and (b) high-grade invasive tumour stage

G3C2. (a) Hsp90 is expressed on the plasma membrane of tumour cells. (b) The signal is more intense at the cell-surface of single malignant cells. Diffuse cytoplasmic and nuclear hsp90 staining is evident in advanced the sections were lightly counterstained with methylgreen malignant carcinoma. Avidin-biotin-peroxidase complex (ABC) method, counterstained with methyl green. (0.5%; 2 min) to visualize nuclei. We studied the tissue samples by assessing the site of staining, the proportion of cell staining (counting at least 1000 cells/sample) and the intensity of staining: weak (+), moderate (++) or strong (+++). among the lines. Reproducibility was assessed by repeating In the negative control slides (absence of immunostaining) the the protein extraction and the SDS-PAGE three times. primary antibodies were replaced with normal mouse serum. Positive controls included human breast cancer (Bio Genex Immunohistochemical studies. Immunohistochemical staining heat shock protein control FG289M). was performed on 3 μm tissue sections by an avidin-biotin- peroxidase complex (ABC) technique using both frozen and Statistical analysis. The significance of differences between formalin-fixed paraffin-embedded tissues. For formalin-fixed different experimental values was assessed by means of paraffin-embedded sections, we applied the microwave oven Anova and Student's t-test. Results were considered to be heating technique, shown to be effective for the retrieval of significant at p-value <0.05. In all cases the same degree of masked epitopes of many antigens. The primary antibodies used significance was obtained with both tests. All statistical were mouse monoclonal antibody AC88, which has a broad manipulations were performed using the SPSS for Windows spectrum of species cross-reactivity against hsp90, including software system (SPSS Inc., Chicago, IL). human hsp90 (30), kindly provided by Dr David O. Toft (Mayo Clinic, Rochester, MN). Briefly, after dewaxing, endogenous Results peroxidase activity was blocked by incubating the sections with methanol containing 3% hydrogen peroxide for 20 min. To explore the potential role of hsp90 family members in the After preincubation with normal goat serum for 20 min a malignant metastatic progression of human colorectal cancer, three-step immunoperoxide procedure was applied: the first we initiated the present study by analyzing the expression step incubated the primary antibodies AC88 at the appropriate profiles of hsp90 family members in various stages of working dilution (1:500, overnight at 4˚C), the second bio- colorectal carcinoma (during the evolution and progression tinylated goat anti-mouse immunoglobulin (Dako, Denmark, of malignancy from pre-malignant lesions to invasive E-433) diluted 1:300 45 min at room temperature, and the carcinomas). Preliminary study showed that not only frozen, third peroxidase-linked avidin-biotin complex (Dako), 1:250 but also formalin-fixed paraffin-embedded sections were diluted, 30 min at room temperature. The peroxidase activity positively stained with the mAbs H9010 and AC88; therefore, was developed by 0.06% H2O2 and 0.03% 3,3'-diamino- the latter were used for the immunohistologic staining study. benzidine tetrahydrochloride for 5 min. After immunostaining, Representative staining patterns are illustrated in Figs. 1-3. As 1169-1178 7/5/08 18:26 Page 1172

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Figure 4. Expression of cytoplasmic hsp90 according to degree of tumour differentiation. The statistically significant difference in the score of cytoplasmic hsp90-immunoreactivity was found between the well-differentiated and poorly-differentiated colorectal adenocarcinoma.

the membrane and cytoplasm, and often in the nuclei of cells (Fig. 2c). The invasive front of tumours displayed strong hsp90 expression at the interphase between tumour and normal tissue (Fig. 3a). Higher magnification of the invasive tumour front displayed strong hsp90 expression in invading tumour cells and also in disseminated single tumour cells expressing nuclear hsp90 (Fig. 3b). Invasive colorectal cancers appeared to be more immunoreactive than superficial tumours. These results indicate that hsp90 might be an important component Figure 3. Hsp90 immunostaining in malignant cells from a high-grade rectal of invasive phenotype of tumour cells. The statistically cancer G2C2 (a) and G1C1 (b). (a) In this case cytoplasmic and to a lesser significant difference in the score of cytoplasmic hsp90- extent nuclear staining was observed. Note the immunoreactivity of cell immunoreactivity was found between the well-differentiated membrane in the invasive front of tumour. (b) Hsp90 immunoreactivity in both cytoplasmic and nuclear regions of nearly all cancer cells. Hsp90 is and poorly-differentiated colorectal adenocarcinoma (Fig. 4). associated with the plasma membrane. Avidin-biotin-peroxidase complex Furthermore, Dukes' C stage groups of colorectal neoplasms (ABC) method, counterstained with methyl green. showed distinct higher positive rates than Dukes' A and B stage groups of tumours. A positive cell surface staining was detectable for hsp90. Membrane expression of hsp90 was also elevated in intracellular hsp90 overexpressing carcinoma shown, immunohistochemically detected hsp90 was mainly cells. In the histologically normal rectal epithelium, we located in the cytoplasm and nucleus of the cancer cells. Hsp90 observed variable degrees of cytoplasmic staining for hsp90 was found in 100% of the specimens. The degree and intensity along the whole length of the crypts (Fig. 2a). of immunoreactivity were uneven, differing from cell to cell Immunoblot analysis of the cytoplasmic protein fraction in the same tumour and among various carcinomas. Cases confirmed that the hsp90 is markedly increased in colorectal with high percentages of immunopositive neoplastic cells cancers [3-6 fold higher by scanning densitometry (Fig. 5B)]. demonstrated intense staining (Fig. 2c). The number of hsp90 The anti-hsp90 antibody H9010 detected three closely spaced positive tumour cells tended to be higher in tumours with bands with the molecular mass of 86, 84 and 82 kDa and detectable lymph node metastases (Figs. 1b, 2c and 3a and b). another at 75 kDa (Fig. 6). The latter form has been found Dispersed fine granules of moderate density, diffusely only in colorectal cancer cells. These data suggested that distributed throughout the cytoplasm, represented a common hsp90N isoform (75 kDa) might be associated with a tumour pattern of immunoreactivity in low-grade tumours (Fig. 1a). formation. Strong immunopositivity was commonly noted in high-grade Two cytosolic isoforms of hsp90, hsp90α (84 kDa) and invasive tumours (Figs. 1b and 2c). The expression of the hsp90ß (86 kDa) are differentially expressed in various stages hsp90 increases as colorectal tumours progress from normal of colorectal carcinoma (Figs. 5 and 6). It is likely that the tissue through various degrees of displastic adenoma to inducible hsp90α isoform is a component of invasive pheno- carcinoma (data not shown). In the central differentiated area type of tumour cells. However, the expression of hsp90ß of the tumour, hsp90 overexpression was detected in both isoform is definitely higher in poorly-differentiated carcinomas 1169-1178 7/5/08 18:26 Page 1173

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Figure 6. Hsp90 levels assayed by Western blotting in the cytoplasmic fractions of colorectal cancer cells. Hsp90 overexpression in pre-neoplastic and neoplastic lesions. Note the hsp90N expression in the cytoplasm of cancer cells. The signal is more intense in the invasive colorectal cancer

stage G2C2 (lane 8). The inducible hsp90α isoform is a component of invasive phenotype of tumor cells. Proteins (20 μg per lane) were electro- phoresed on a 12% polyacrylamide gel and immunoblotted with the H9010 antibody at the dilution of 1:500 and AP-cojugated goat anti-mouse IgG at the dilution 1:1000. Immunoreactive proteins were visualized with an NBT/BCIP (Gibco). Numbers refer to the relative molecular mass in kilodaltons of the standard proteins (S). Lanes 1 and 3, normal mucosa; 2

and 4, preneoplastic lesions; lanes 5-9, colorectal cancer, G2C2, G1C2, G2B2,

G2C2, G3C2 (interstitial type).

Figure 5. (A) Western blot analysis of hsp90 expression in cytoplasmic fraction of colorectal cancer cells. Differential stage-dependent expression of cytosolic hsp90 isoforms. Hsp90 migrated as three closely spaced bands (86, 84 and 82 kDa). Two cytosolic isoforms of hsp90, α and ß, are differentially expressed in various stages of colorectal carcinoma. A higher level of hsp90α isoform (84 kDa) and hsp90ß (86 kDa) seems to be closely correlated with the colorectal cancer progression. Hsp90ß isoform expression was significantly higher in poorly-differentiated carcinomas than in well- differentiated colorectal cancers. Cytosolic extracts were prepared as described in Materials and methods. Proteins (20 μg per lane) were electro- phoresed on a 12% polyacrylamide gel and immunoblotted with the anti- hsp90 antibodies H9010 at the dilutin of 1:500 and AP-conjugated goat Figure 7. Western blot analysis of hsp90 expressed on the plasma membrane anti-mouse IgG at the dilutin 1:1000. Visualization of signals was performed of colorectal cancer cells. Indicated by intense band, high amounts of hsp90 by NBT/BCIP solution. The data shown are from one representative set of were present in plasma membrane of poorly-differentiated adenocarcinoma three independent experiments. Molecular mass markers are presented on the stage G3C2. We did not distinguish between hsp90α and ß in the present left (S). The position of the hsp90 is indicated by the arrowheads. The analyses of different types of lesion. Membrane expression of hsp90N arrowhead indicates an hsp90N (75 kDa) isoform; lanes 1-4, normal (75 kDa) is significantely higher in poorly-differentiated cancer. Proteins (80 μg per lane) were subjected to SDS-PAGE and probed with anti-hsp90 mucosa; lanes 5-9, colorectal cancer G1B2, G1C2, G3C2, G1B1, G1C2. (B) Densitometric quantification of the expression of hsp90. The intensity of the antibodies H9010 and AP-cojugated goat anti-mouse IgG, all at a dilutin of bands was determined densitometrically. Significant increase of the hsp90 1:500. Immune complexes were detected using the substrate NBT/BCIP in cytosolic fractions of colorectal cancer is evident. K, normal tissue (Gibco). Left margin, molecular mass standard (s). The position of the hsp90 (pattern nos. 43, 51-53); T, tumour tissue (pattern nos. 48, 54-56, 61). is indicated by the arrowheads. Lanes 1 and 2, normal mucosa; lanes 3-7, colorectal cancer G1B1, G2B2, G1C2, G1C2, G3C2 (Dukes' stage and degree of differentiation).

than in well-differentiated colorectal cancers, suggesting an involvement of hsp90ß in the inhibition of cancer cell differ- as evidenced by the presence of abnormal clones of cells in entiation. The expression of hsp90 in poorly-differentiated morphologically normal mucosa. The analysis indicated a colorectal cancer with metastasis was definitely higher than hsp90 overexpression in pre-neoplastic and neoplastic lesions. that in well-differentiated colorectal cancer without metastasis Additionally, we identified the hsp90 at the plasma membranes (Fig. 5). Surprisingly, high expression of hsp90 are typically of colorectal cancer cells. Hsp90 was originally thought to found in ‘normal’ tissue surrounding tumours. The ‘normal’ be ubiquitously expressed as a cytoplasmic protein whose tissue (Fig. 6, lanes 2 and 4) shows a higher level of hsp90α/ß, function was to capture folding intermediates to prevent protein 1169-1178 7/5/08 18:26 Page 1174

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misfolding and aggregation and to facilitate proper expression of hsp90 in poorly-differentiated colorectal cancer refolding. with metastasis was definitely higher than that in well- We found by analysis that hsp90 is associated with the differentiated colorectal cancer without metastasis, indicating plasma membrane of colorectal carcinoma cells. The most that up-expression of hsp90 is likely to have some prominent band of hsp90 was detected in the case of poorly- relationship with progression, invasion and metastasis of

differentiated colorectal adenocarcinoma (G3C2), in general, colorectal cancer. Additionally, we support the idea that the more aggressive and with worse prognosis than moderately high hsp90 expression may lead to the identification of the and well differentiated tumours (Fig. 7, lane 7). The same earliest pathological features initiating colorectal tumouri- protein is present in trace amounts in corresponding non- genesis. Furthermore, overexpression of the hsp90 protein is neoplastic colonic mucosa excluding the muscle layer, from also a feature of pre-invasive colorectal carcinomas. In these the same patient (Fig. 7, lane 2). Membrane expression of cases greater staining for hsp90 protein has been detected in hsp90 was also elevated in normal mucosa tissue collected by the invasive margins of the tumours. This evidence suggests dissection from macroscopically normal areas from patients that hsp90 family are likely to be one of the critical players in to whom colorectal cancer tissues were surgically removed colorectal cancer both in the initiation stage and during (Fig. 7, lane 1). This might in part explain the appearance of invasion and metastasis. hsp90 at the surface of normal mucosa cells. Essentially, there are indications that changes in However, we did not distinquish between hsp90α and ß proliferation-related genes such as hsp90 may occur in a in the present analyses of plasma membranes (Fig. 7). progressive fashion and that this may be reflected in the Moreover, it has been shown that the anti-hsp90 monoclonal association of hsp90 overexpression with tumour grade and antibody H9010 cross-reacted with additional hsp90-like the degree of differentiation. Thus, we have attempted, and proteins (Fig. 7). hopefully achieved herein, a conceptual as well as a factual synthesis of the biological processes of invasion, Discusion proliferation, and metastasis, by linking the biological processes of cancer invasion and secondary spread with cell Increasing evidence indicates that colorectal cancer is a proliferation and its deregulation. It seems reasonable genetically heterogeneous and complicated disease (31-33). therefore to regard that hsp90s contribute significantly to In the invasion and metastasis of colorectal cancer many genes the invasive propensity of cancer cell and are powerful have been identified as being involved in proteolysis, modulators of metastatic behaviour. adhesion, angiogenesis and cell growth (34). The hsp90 Although a great deal of information is currently available molecular chaperone catalyses the final activation step of regarding the structure of the hsp90 complex and its regulation, many of the most important regulatory proteins of eukaryotic the involvement of this protein in biological processes such cells (2). They participate in almost every remodeling event as proliferation and differentiation requires further investi- involving larger protein complexes, including signalling, gation. Nevertheless, it should be remembered, that the hsp90 transcription, cell division, migration, and differentiation (2). functions in certain aspects of cellular differentiation. Yamada In transformed cells, numerous mutated and overexpressed et al (20) found that hsp90 showed remarkably high expression proteins rely on the hsp90 protein folding machinery for in undifferentiated human embryonal carcinoma cells. As a tumour progression. The heat-shock proteins hsp70 and hsp90 result, c-src tyrosine kinase was associated with hsp90 in induced by cell stress are expressed at high levels in a wide undifferentiated cells. These results suggest that hsp90 may range of tumours and are closely associated with a poor play important roles on cell differentiation through the control prognosis and resistance to therapy. The increased of tyrosine kinase activities (20). In addition, differentiation of transcription of hsps in tumour cells is due to loss of p53 embryonal carcinoma cells is paralleled by the induction of function and to higher expression of the proto-oncogenes hsp90ß (37) and a non-identified isoform of hsp90 (38). Hsp90 HER2, c-Myc, k-ras and other genes is crucial to binds to a wide variety of cellular proteins and is thought to tumourigenesis (35). The hsp family members play play a role in a number of physiological processes including overlapping essential roles in tumour growth both by chromatin condensation (39), protein synthesis (40), protein promoting autonomous cell proliferation and by inhibiting folding (41) and protein trafficking (42). More importantly, death pathways (36). By chaperoning key proteins such as it has been shown that hsp90 functions in the regulation of Raf-1, Akt, survivin and hTERT, hsp90 regulates signalling the cell cycle (43,44), growth and differentiation processes pathways necessary for the growth, survival and limitless (45,46). In this respect, hsp90 may be involved in one or more replicative potential of most tumours. Important elements of of these phenomena during development of the colorectal the apoptotic pathways are also regulated by hsp90 (36). The cancers. variety of functions attributed to hsp90 proteins suggest the Hsps and the heat-shock response have been shown in a potential role of hsp90 in colorectal carcinogenesis. number of settings to protect against apoptosis (14,16,17,22). The important finding of this study is that elevated Noteably, hsp90 is at a crossroad for multiple signalling expression of hsp90α/ß as well as the hsp90N isoform pathways (6,7). It is becoming clear that hsp90 chaperoning commonly observed in colorectal cancers analyzed are often is not only essential to a variety of signal transduction associated with more invasive and metastatic phenotypes. It pathways, but is also critical for proper folding, stabilization should be pointed out that there was a definite correlation and trafficking of an expanding list of proteins. Three between expression of hsp90 and development and components of the MAP kinase signalling system (src, raf metastasis of colorectal cancers. 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protein that is the mammalian homolog of the yeast cell cycle genome rearrangements are frequent in tumour cells, one control protein cdc37 (47). This suggests that binding to might speculate that among possible rearrangements, gene hsp90 is in some way related to the transforming activity of amplifications are often involved in colorectal tumouri- src. These heterocomplexes are formed by a multiprotein genesis. Difficulties, especially in the context of multiple chaperone machinery consisting of at least four ubiquitous genomic rearrangements, are encountered in ascertaining proteins, hsp90, hsp70, p60 and p23 (47). Hsp90 affects both whether the overexpression of a given gene is the direct the stability and localization of the p53 mutants (48). Using consequence of a change in its copy number or of the a temparature-sensitive mutant of p53, Sepehrnia et al (49) overexpression of non-amplified alleles. Jolly et al (74) have found that at the nonpermissive temperature, p53 mutant was shown in human A431 carcinoma cells, that increased localized in the cytoplasm and in a heterocomplex with expression of the hsp90ß gene is due at least in part, to gene hsp90, whereas at the permissive temperature, the p53 was amplification. The amplification of the hsp90ß gene may nuclear and not immunoadsorbed with hsp90. Thus, as for involve an adjacent oncogene, such as the pseudo-gene v-Ki- the GR, hsp90 affects both the stability and localization of ras1, which maps to the Gp12-6p11 region (75). Therefore, the p53 mutants. abnormalities of this gene may appear early in tumour Hyperactivation of growth promoting target genes of development or in late stages of progression of colorectal ß-catenin-TCF signalling including c-myc may promote the cancers. onset of oncogenesis. The c-myc proto-oncogene encodes Hsp90 action on the cell surface might be a wide-ranging a ubiquitous transcription factor involved in the control of phenomenon. Hsp90 has been implicated in peptide antigen cell growth and differentiation and is implicated in inducing presentation to macrophages and dendritic cells (76). This colorectal tumourigenesis (32). Understanding the function suggests the possibility that hsp90 chaperones a broad array of c-myc and its role in cancer depends upon the identi- of proteins and peptides in the extracellular space that act in a fication of c-myc target genes. The findings of Teng et al variety of cellular processes. (50) that c-myc directly activates hsp90α transcription In addition to promoting signalling events, hsp90 also suggest that by induction of hsp90α c-myc may control the functions in the processes of cell migration. Invasion and activity of multiple signal pathways involved in cellular metastasis are exceedingly complex processes, and their transformation. genetic and biochemical determinants remain incompletely Although not as extensively analyzed, hsp90α expression understood (77). An understanding of the means by which in particular is crucial in cancer (51,52). It is noteworthy that tumour cells achieve metastatic dissemination is obviously of hsp90 has a significant role in triggering complex biological crucial importance for successful treatment of the disease and processes such as cell proliferation, differentiation or for evolving possible strategies for the prevention of secondary transformation (53). In fact, both hsp90 and grp94 are spread. Although in the clinical context the events associated frequently up-regulated in tumour cells experiencing various with the generation of metastatic and drug resistant phenotypes of stress, such as acidic pH, a scarcity of nutrients, and types within the tumour contribute significantly to failure of fluctuations of oxygen supply (54). Thus, constitutively treatment, these events are still poorly understood at the elevated levels of hsp90 were found in ras-transformed cells biochemical and molecular biological level. Current post- (51), other malignant cell lines (55-57), acute leukemias genomic strategies are being applied to identify genes and (52,58), melanomas (59), gastrointestinal cancers (60), their encoded proteins that function in tumour invasion. ovarian cancers (61) and in pancreatic and endometrial Eustace et al (78) showed that the hsp90α isoform, but not carcinomas (62,63). Grp94 was found to be up-regulated in hsp90ß, is expressed extracellularly on fibrosarcoma and colon adenocarcinoma (64) and in large, radiation-induced breast cancer cells: hsp90α interacts with the matrix metallo- mouse fibrosarcomas (65). Both hsp90 and grp94 are proteinase 2 (MMP2) outside the cell and promotes MMP2 overexpressed in human breast cancer (66-68) where activation, which is critical for tumour invasiveness. This overexpression of hsp90α is usually associated with poor finding extends the multiplicity of roles assigned to the hsp90 prognosis (69). Complementing these changes, the down- family to a new function outside the cell. The cytosolic regulation of hsp90ß has been observed in the invasive and molecular chaperone hsp90 is now shown to promote tumourigenic BC-61 subline of 8701-BC breast carcinoma maturation of the extracellular MMP2 required for the invasive cells (70). A higher level of heat-shock proteins, particularly nature of cancer cells (79). Therefore, the overexpression of hsp90α seems to be closely correlated with the overall hsp90 in tumour cells stimulates the metastatic process. proliferative potential of malignant cells. This is in perfect Malignant tumours are known to be heterogeneous with agreement with the results described above and these data respect to the expression of biochemical markers, degree of strongly support the idea that hsp90 is involved in the differentiation, and immunogenicity. It is now generally development of a colorectal carcinomas. accepted that they are not composed of cells of equal metastatic Elevated levels of heat-shock proteins may participate in potential but contain a variety of subpopulations with widely the reorganization of chromatin structure, help in the main- differing invasive and metastatic capabilities, proposing that tenance of estrogen-dependent growth (71,72) and confer a tumour development involved traverse through different significant advantage on tumour cells to survive in a hostile stages where the neoplasm could be seen to acquire cellular and environment. Increased amounts of hsp90 may also lead to functional features that could be used to define its progression. an increased drug resistance of certain tumours (73). Thus, there will exist subpopulations with diverse biological Following the idea that the changes in gene structure properties, including metastatic potential, depending on the and/or in the level of gene expression as a consequence of degree of progression undergone by the tumour. Although our 1169-1178 7/5/08 18:26 Page 1176

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